我们感谢艾格尼丝的Viale博士和MSKCC基因组学核心实验室提供技术援助。此协议是从杰弗里比尼癌症研究中心和农民家庭基金会的支持下开发的。

1。 DNA和试剂制备注意：该协议描述了24个样本（ 例如，12种肿瘤/正常对）的同时进行处理和分析，但可以适用于更小和更大的批次。 DNA样本可能来自FFPE或新鲜冷冻组织，细胞学标本，或血液。通常，这两种肿瘤和正常组织的来自同一患者将异形在一起，以便从遗传多态性区分的体细胞突变。该协议将立即开始DNA提取以下。 <ol><li>等分试样50-250纳克（250纳克推荐）每个样品中提取的DNA，稀释在1x的Tris-EDTA（pH值8.0）缓冲液中，以50微升的最终体积，为单独的Covaris圆底试管中。 </li><li>剪切力对Covaris E220仪器的DNA在以下剪切设置：360秒，10占空因数，175画中画，每爆裂200次。 </li><li>解冻AMPure XP珠（ 表2）至室温。解冻所有BUffers和酶在冰上之前的适当步骤。 </li></ol> 2。文库制备 - 完维修模块<ol><li>使用每个样品以下几卷准备结束维修师傅混在一个无菌的离心管：10微升10X NEBNext结束修复反应缓冲液（ 表2），5微升NEBNext结束修复酶混合物（ 表2），35微升无菌核酸游离H 2 O。 </li><li>分装50μl的每个剪切的DNA到96孔板的单独的孔中。加入50微升的准备结束维修师傅组合到每个反应。在20℃下孵育在热循环仪进行30分钟</li></ol> 3。结束后的修复清理<ol><li>添加AMPure XP珠（ 表2）的2倍体积（200微升），以每个样品，并使用多道移液器轻轻地吸取了整个音量向上和向下10倍。在室温下孵育15分钟。 </li><li>广场上的磁性支架（ 表2 </STRONG&gt;），在室温下15分钟，直到样品出现明显的。 </li><li>轻轻取出并丢弃上清液，注意不要打扰珠。一些液体可能会留在井中。 </li><li>与在支架管，轻轻地加入200微升新鲜制备的80％乙醇中的每个样品，孵育板在室温下持续30秒。小心，通过吸管除去乙醇。 </li><li>重复步骤5，共2乙醇洗涤。确保所有的乙醇已被删除。从磁性支架取出，放于干燥室温下5分钟。 </li><li>重悬干燥珠粒与44.5微升无菌H 2 O。轻轻地，移液管整个音量上下10X充分混合。确保珠粒不再连接到井的侧面。 </li><li>孵育再悬浮珠粒在室温下持续2分钟。将磁静置5分钟，直到样品出现明显的。 </li><li>轻轻地转移42微升含有样品到一个新的好清楚上清液。 该程序可以安全地停在这个STEP并储存在-20℃下7天的样品。要重新启动，继续操作前解冻冷冻样本置于冰上。 </li></ol> 4。文库制备 - DA-泰陵模块<ol><li> 5微升10倍NEBNext大拖尾反应缓冲液（ 表2），3微升（3&#39;-5&#39;外切）Klenow片段（ 表2：使用每样以下几卷准备大拖尾预混液在无菌的离心管）。 </li><li>加入8μL准备好的大拖尾主结构，每孔含42微升的最终修复的DNA。在37℃下孵育在热循环仪进行30分钟</li><li>执行后大拖尾清理：用无菌H 2 O至33.75微升，最终体积2倍体积（100微升）的珠子，但重悬，重复步骤3.1-3.8。 该程序可以安全地停止在该步骤中，保存于-20℃下进行长达7天的样品。要重新启动，解冻冷冻样品上，然后再继续冰。&lt;/ LI&gt; </ol> 5。文库制备 - 接头连接模块<ol><li> 10微升5倍NEBNext快速连接反应缓冲液（New England Biolabs公司， 表2），5微升快速T4 DNA连接酶（ 表2）：使用每个样本以下几卷准备结扎预混液在无菌的离心管。 </li><li>加入15微升连接主结构，每孔含DA尾的DNA。 </li><li>加入1.25微升适当的25微米NEXTflex条形码适配器（ 表2），每孔。每个样品应接受一个单独的条形码适配器。在20℃下孵育在热循环仪进行15分钟</li><li>在无菌H 2 O的使用珠1X体积（50μL）重复步骤3.1-3.8，但重悬至50微升，最终体积：执行后接头连接清理。 </li><li>对于第二个1X卷（50微升）清理重复步骤3.1-3.8，但重悬在无菌H 2 O至一23微升最终体积。 该程序可以安全地停止在该步骤中，保存于-20℃下进行长达7天的样品。要重新启动，继续操作前解冻冷冻样本置于冰上。 </li></ol> 6。文库扩增<ol><li>准备KAPA高保真主结构中使用下列卷每个样品的无菌微量离心管：25微升2倍KAPA高保真HS马币（ 表2），每个100μM引物1和2（ 表3），1微升。 </li><li>加27微升主混合物到每个孔中含有的连接产物。设置吸至50μl，轻轻吸取整个音量上下10倍。 </li><li>放置在热循环仪中进行以下PCR循环：45秒，在98℃初始变性，10个循环15秒，在98℃，30秒，60℃，30秒，72℃，1分钟的最终延伸在72°C。 </li><li>进行扩增后的清理：使用重复步骤3.1-3.8AMPure XP珠的1X卷（50微升），重悬在无菌水中，以30微升的最终体积。 </li><li>量化每个库的根据制造商的说明书，使用量子比特宽范围测定（Life Technologies公司， 表2）的浓度。 </li></ol> 7。罗氏NimbleGen SeqCap的EZ图书馆杂交，洗涤和扩增<ol><li>解冻普及和指数寡核苷酸阻断剂（ 表3），SeqCap的EZ图书馆和NimbleGen的捕捉组件（COT的DNA，2个杂交液，杂交和成分A， 表2），在冰上。所有的寡核苷酸阻断剂都存放在1毫米工作股。我们SeqCap的EZ图书馆是捕获探针涵盖的279癌症相关基因的所有蛋白质编码外显子，但是可以使用其他自定义和目录设计定制设计的游泳池。 </li><li>准备对应于所述特定条形码的广告索引寡核苷酸阻断剂的1 mM的池在文库制备用于aptor序列。结合1μl的各阻断剂，和涡流。 </li><li>在一个新的无菌的1.5 ml离心管中，加入5微升1mg/ml的COT的DNA，2微升1毫米通用阻滞剂和2微升指数寡核苷酸阻断剂的1 mM的游泳池。 </li><li>池24条形码库到一个单一的反应。一共有1-3微克汇集，条形码序列库（上述制备）加入到捕捉混合物中。对于24个样品，每个样品100纳克建议。当汇集肿瘤和匹配的正常样本，我们建议增加更多的肿瘤库（ 如每个肿瘤和正常每50毫微克100毫微克），使它们测序，以更深入的报道。 </li><li>关闭该管帽，使15-20的孔中具有18-20 G或更小的针帽的顶部。 Speed​​ Vac中扩增样本库/ COT DNA /在高温（60℃）的DNA真空浓缩寡核苷酸。 </li><li>一旦完全干燥下来，补充水分与7.5微升2倍的有杂交zation缓冲液和3微升杂交组分A的管现在应包含以下内容：5微克（COT）的DNA，可变微克放大样品库1000皮摩尔通用和指数寡核苷酸，7.5微升2×杂交缓冲液，在3微升杂交A组份10.5μl总体积。 </li><li>覆盖在帽上的孔与一小块实验室胶带。旋涡样，持续10秒，并以最大速度离心10秒。 </li><li>孵育样品在95℃下10分钟以使DNA变性，然后离心以最大的速度持续10秒在RT。 </li><li>在新0.2毫升的PCR条管，准备SeqCap的EZ图书馆捕获探针和2.25微升无菌无核酸酶的H 2 O的的2.25微升等分，并添加7.7步的内容。轻轻吸取整个音量上下10倍。 </li><li>孵育在热循环仪在47℃下进行48-96小时。设置和维护的热循环仪的盖子温度为57°C。 </li><li>折伞在NimbleGen的SeqCap的EX图书馆SR用户指南V3.0（ 表2）进行洗涤和捕获的DNA的回收第6章低步骤。 </li><li>如下在罗氏NimbleGen协议所捕获的DNA进行了以下修改的放大的第7步： <ul type="disc"><li>使用2个KAPA高保真聚合酶代替的Phusion高保真PCR扩增预混试剂（ 见表2） </li><li>使用100μM的引物1和引物2在表3中列出。 </li><li>运行以下PCR条件：在98℃起始变性30秒，11个循环的98℃10秒，60℃30秒，72℃30秒，72℃最后延伸5分钟。保持在4°C。 </li></ul></li><li>量化，根据制造商的说明使用量子比特的高灵敏度测定（ 表2）的扩增捕获的DNA的浓度。 </li><li>分析使用安捷伦Bio捕获DNA的质量分析器的DNA芯片HS（ 表2）按照制造商的说明。 <ul type="disc"><li>将PCR产率至少应为100毫微克（范围100-1000毫微克）。 </li><li>的平均片段长度应该是150-400个碱基对之间。 </li></ul></li></ol> 8。 Illumina公司的Hi-序列测序<ol><li>稀释扩增的DNA捕获到下午2点，并装入样品上的Illumina的HiSeq 2000流动池的单一车道。根据制造商的说明书序列配对末端75个碱基对的读取。根据我们的经验，一个车道就足以产生每个样品500-700X唯一的序列覆盖整个279基因的24个样品池。 </li><li> Illumina公司的软件（实时分析）将高分辨率的图像转换为集群强度为基础的呼叫和质量分数。使用CASAVA根据条形码的身份去复用数据，并生成单个FASTQ文件，每个样品。 </li></ol> 9。数据分析ysis <ol><li>从序列的3&#39;端修剪接头序列读取使用Cutadapt（FASTQ文件<a href="http://code.google.com/p/cutadapt/">http://code.google.com/p/cutadapt/</a> ）。最小搭接长度（-O）为3 1％的错误率（-E）。最低基数长度保持配对的修剪是25后读取。 </li><li>排列顺序来读取使用Burrows-惠勒调校工具（BWA）12参考人类基因组hg19。执行后处理步骤，包括根据自己的标准最佳实践（使用基因组分析工具包（GATK）重复的标记，当地多序列调整和基本质量得分重新校准<a href="http://www.broadinstitute.org/gatk/guide/topic?name=best-practices">http://www.broadinstitute.org/gatk/guide/topic？名称=最佳实践</a> ）13。当分析来自同一患者的多个样品（ 例如，匹配的肿瘤和正常），本地多序列调整应的perforMED联合。这些后处理步骤的输出是对每个样品，其中包含的所有序列，质量和取向信息，可直接加载到结合基因组浏览器（IGV），用于读取和序列变体14的可视化的单独的BAM文件。 </li><li>使用皮卡德工具计算序列的性能指标（ <a href="http://picard.sourceforge.net/">http://picard.sourceforge.net/</a> ）。信息化指标包括对齐率，片段大小分布，GC含量相关报道的偏见，对目标的捕获特异性，PCR重复率，库的复杂性，和平均目标覆盖。代表值被显示在图1。等位基因频率在使用DepthOfCoverage从GATK计算常见多态性位点被用来从无关的DNA污染监测，并确保匹配样本来自同一患者。 下面的计算分析依赖于匹配的存在肿瘤和正常样本进行体细胞的遗传改变的检测。无与伦比的肿瘤也可以使用一个单独的正常对照样品进行分析，但大多数的体细胞突变不能容易地从继承的序列变体区分开来。 </li><li>利用体细胞突变来电，如muTect 15，SomaticSniper 16，或Strelka 17各肿瘤对正常体细胞调用单核苷酸变异（SNVs）。我们使用muTect具有修饰的过滤标准，允许与正常样品中的低（非零）等位基因计数只要等位基因频率是至少5倍的程度，在肿瘤的变种。变种循环地称为在正常的DNA面板也将被移除排序或排列的可能系统性文物。每个SNV通过过滤器，然后仔细审查使用IGV。 </li><li>叫躯体插入缺失对每个肿瘤对正常使用的算法如SomaticIndelDetector 13，Dindel 18，或19 SOAPindel </sup&gt;。我们使用SomaticIndelDetector用以下标准：至少10％的等位基因频率与至少3支持读取，或者至少2％的等位基因频率与至少10支撑的读取。我们允许插入缺失与正常样品中的低（非零）等位基因计数只要等位基因频率是至少5倍更大的肿瘤。插入缺失称为在正常的DNA面板被去除，如上述。每个插入缺失通过过滤器，然后仔细审查使用IGV。 </li><li>从序列覆盖在目标外显子外推套数状态。对于每一个样本，为所有目标外显子，回归了GC比例进行平均序列覆盖的黄土正常化。减去黄土配合和添加样品的全覆盖中位数调整，非标准化的覆盖值。计算比例，在所有目标外显子的肿瘤对正常正规化序列覆盖率。如果没有匹配的正常样本和/或降低噪声，其他与性别相匹配的二倍体正常对照缺乏种系拷贝数变异体也可使用。确定体拷贝数得失的基础上的覆盖率增加或减少。 </li><li>调用涉及癌基因，其中至少一个基因组中断点落在范围内或附近的基因组的目标间隔体细胞重排。建议的算 ​​法包括GASV 20，Breakdancer 21，CREST 22，和dRanger 23。染色体内的倒位，缺失，和串联重复可以根据支撑的相对位置和方向读取不和谐对区别开来。所有考生重排，应人工审查使用IGV。我们建议实验验证通过在假定的重排断点PCR和Sanger测序。 </li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+somatic+point+mutations+in+impure+and+heterogeneous+cancer+samples.">Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples.</a> Nat. Biotechnol. 31 (3), 213-219 (2013).</li><li>Larson, D. E., Harris, C. C., Chen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SomaticSniper:+identification+of+somatic+point+mutations+in+whole+genome+sequencing+data.">SomaticSniper: identification of somatic point mutations in whole genome sequencing data.</a> Bioinformatics. 28 (3), 311-317 (2012).</li><li>Saunders, C. T., Wong, W. S., Swamy, S., Becq, J., Murray, L. J., Cheetham, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strelka:+Accurate+somatic+small-variant+calling+from+sequenced+tumor-normal+sample+pairs.">Strelka: Accurate somatic small-variant calling from sequenced tumor-normal sample pairs.</a> Bioinformatics. 28 (14), 1811-1817 (2012).</li><li>Albers, C. A., Lunter, G., Macarthur, D. G., Mcvean, G., Ouwehand, W. H., Din del Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+indel+calls+from+short-read+data.">Accurate indel calls from short-read data.</a> Genome Res. 21 (6), 961-973 (2011).</li><li>Li, S., Li, R., Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+identification+of+indels+from+short+paired+reads.">Efficient identification of indels from short paired reads.</a> Genome Res. 23 (1), 195-200 (2013).</li><li>Sindi, S., Helman, E., Bashir, A., Raphael, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+geometric+approach+for+classification+and+comparison+of+structural+variants.">A geometric approach for classification and comparison of structural variants.</a> Bioinformatics. 25 (12), 222-230 (2009).</li><li>Chen, K., Wallis, J. W., Mclellan, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BreakDancer:+an+algorithm+for+high-resolution+mapping+of+genomic+structural+variation.">BreakDancer: an algorithm for high-resolution mapping of genomic structural variation.</a> Nat. Methods. 6 (9), 677-681 (2009).</li><li>Wang, J., Mullighan, C. G., Easton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CREST+maps+somatic+structural+variation+in+cancer+genomes+with+base-pair+resolution.">CREST maps somatic structural variation in cancer genomes with base-pair resolution.</a> Nat. Methods. 8 (8), 652-654 (2011).</li><li>Drier, Y., Lawrence, M. S., Carter, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+rearrangements+across+cancer+reveal+classes+of+samples+with+distinct+patterns+of+DNA+breakage+and+rearrangement-induced+hypermutability.">Somatic rearrangements across cancer reveal classes of samples with distinct patterns of DNA breakage and rearrangement-induced hypermutability.</a> Genome Res. 23 (2), 228-235 (2012).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10 (1), 57-63 (2009).</li><li>Roychowdhury, S., Iyer, M. K., Robinson, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+oncology+through+integrative+high-throughput+sequencing:+a+pilot+study.">Personalized oncology through integrative high-throughput sequencing: a pilot study.</a> Sci. Transl. Med. 3 (111), 111ra121 (2011).</li><li>Kerick, M., Isau, M., Timmermann, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+high+throughput+sequencing+in+clinical+cancer+settings:+formaldehyde+fixed-paraffin+embedded+(FFPE)+tumor+tissues,+input+amount+and+tumor+heterogeneity.">Targeted high throughput sequencing in clinical cancer settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity.</a> BMC Med. Genomics. 4, 68 (2011).</li></ol>

Berger博士已收到的咨询费和科研经费从基础医学，癌症诊断公司。

我们的影响分析产生的高定位速度，较高的目标速度，目标高覆盖，高灵敏度检测基因突变，插入缺失和拷贝数变化。我们已经证明我们的影响分析的能力，同时从新鲜冰冻的DNA序列和归档低的DNA输入FFPE样品。通过执行主要癌症相关的基因有针对性的外显子测序，可以达到非常深的序列覆盖率为这些最关键的基因的外显子从而最大限度地检测低频率突变的能力。使用条形码和复用的，可实现更高...

在关键的癌基因和肿瘤抑制基因的“驱动器”肿瘤的遗传事件的识别起着许多癌症1的诊断和治疗的重要作用。利用大规模并行“下一代”测序大规模的研究工作已经启用了很多这样的癌症相关的基因，近年来2的鉴定。然而，这些测序平台通常需要大量的DNA从新鲜冰冻组织中分离，从而构成一个重大的限制在从保存组织，如福尔马林固定，石蜡包埋（FFPE）的肿瘤样品表征和分析DNA突变。改进的努力，高效，可靠地从FFPE肿瘤样本的特征“可操作的”基因组信息将使以前存入银行的标本进行回顾性分析，并进一步鼓励个性化的方法对癌症的管理。 传统上，分子ðiagnostic实验室一直依赖耗时，低通量的方法，如Sanger测序及实时荧光定量PCR进行DNA突变分析。最近，利用复用PCR或质谱基因分型高通量方法已被开发来调查在关键的癌基因3-5复发性体细胞突变。这些方法，但是，只有预先指定的“热点”的突变进行了检测，使得它们不适合用于检测失活突变的肿瘤抑制基因受到限制。大规模并行测序提供了这些策略的几个优点，包括审问整个外显子为双方共同和稀有突变的能力，才能看到其他类基因组改变，如拷贝数收益和损失的能力，异构样品6，在更高的检测灵敏度7 。全基因组测序为代表的突变的发现的最全面的方法，虽然它是相对的LY昂贵，即被用于数据分析和存储大量的计算需求。 对于临床应用，其中的基因组中的仅一小部分可能是临床利益，在测序技术2特别的创新已经变革。首先，通过基于杂交的外显子捕获，可以分离DNA对应键与癌症相关的基因突变有针对性的分析8。第二，通过分子条形码（ 即 DNA序列的6-8个核苷酸长）的结扎，可以有数百个每个测序运行样品并充分利用大规模并行测序仪器10不断增加的容量的优点。合并后，这些创新使肿瘤可为异形更低的成本和更高的吞吐量，更小的计算需求11。进一步，通过重新分配序列覆盖，只有那些基因最关键的特定应用，可以achie已经较大的测序深度为低等位基因频率较高的事件检测灵敏度。 在这里，我们描述了我们的影响分析（中可操作的癌症靶点整合突变图谱），使用自定义的寡核苷酸捕获所有蛋白质编码外显子和选择的279键的癌症相关基因的内含子（它利用外显子捕获的条形码序列库池通过杂交表1 ）。这一战略使基因突变，插入缺失，拷贝数改变的识别，并选择涉及这些279个基因的结构重排。我们的方法是与DNA来自新鲜冷冻和FFPE组织以及细针抽吸等细胞学标本中分离出兼容。

<table border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody> 
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext End Repair Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6050L</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext dA-Tailing Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6053L</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEBNext Quick Ligation Module</td>
			<td style="width:247px;">New England Biolabs</td>
			<td style="width:137px;">E6056L</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agencourt AMPure XP</td>
			<td style="width:247px;">Beckman Coulter Genomics</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NEXTflex PCR-Free Barcodes &#8211; 24</td>
			<td style="width:247px;">Bioo Scientific</td>
			<td style="width:137px;">514103</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HiFi Library Amplification Kit</td>
			<td style="width:247px;">KAPA Biosystems</td>
			<td style="width:137px;">KK2612</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">COT Human DNA, Fluorometric Grade</td>
			<td style="width:247px;">Roche Diagnostics</td>
			<td style="width:137px;">05 480 647 001</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NimbleGen SeqCap EZ Hybridization and Wash kit</td>
			<td style="width:247px;">Roche NimbleGen</td>
			<td style="width:137px;">05 634 261 001</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SeqCap EZ Library Baits</td>
			<td style="width:247px;">Roche NimbleGen</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">QIAquick PCR Purification Kit</td>
			<td style="width:247px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Qubit dsDNA Broad Range (BR) Assay Kit</td>
			<td style="width:247px;">Life Technologies</td>
			<td style="width:137px;">Q32850</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Qubit dsDNA High Sensitivity (HS) Assay Kit</td>
			<td style="width:247px;">Life Technologies</td>
			<td style="width:137px;">Q32851</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agilent DNA HS Kit</td>
			<td style="width:247px;">Agilent Technologies</td>
			<td style="width:137px;">5067-4626, 4627</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Agilent 2100 Bioanalyzer</td>
			<td style="width:247px;">Agilent Technologies</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Covaris E220</td>
			<td style="width:247px;">Covaris</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Magnetic Stand-96</td>
			<td style="width:247px;">Ambion</td>
			<td style="width:137px;">AM10027</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Illumina Hi-Seq 2000</td>
			<td style="width:247px;">Illumina</td>
			<td style="width:137px;"> </td>
			<td style="width:79px;"> </td>
		</tr>
	</tbody>
</table>

detecting somatic genetic alterations in tumor specimens by exon capture and massively parallel sequencing

努力发现和调查的关键致癌基因突变已被证明有价值的，以方便对癌症患者进行相应的处理。高通量的建立，大规模并行“下一代”测序已经资助了许多这样的突变的发现。以提高该技术的临床和平移工具，平台必须是高通量，成本效益，并用福尔马林固定，石蜡包埋（FFPE）组织样品可能产生少量的退化或受损的DNA兼容。在这里，我们描述了随后靶向外显子用于癌症相关的突变在新鲜冰冻和石蜡包埋的肿瘤通过大规模平行测序检测的基于杂交的捕获条形码和复用DNA文库的制备。这种方法使序列突变的识别，拷贝数变化，并选择涉及所有目标基因的结构重排。针对外显子测序提供吨他同样拥有高通量，低成本，和深序列覆盖率，从而赋予高灵敏度检测低频率突变。

使用对应于279癌基因的所有蛋白质编码外显子探针24条形码序列库（12肿瘤对正常）一池被抓获，并测序为2×75 bp的读取上一个的HiSeq 2000流动池的单一车道。肿瘤和正常库汇集以2:1的比例。冷冻肿瘤DNA样品的样品池的性能指标示于图1，其中包括对应率，片段大小分布，对靶标捕获的特异性，和平均目标覆盖范围。例如体细胞突变，插入，缺失和拷贝数改变的示于图2-4中 。...

Watch this Scientific Journal Video about Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing at JoVE.com

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

我们描述条形码DNA文库和随后的杂交为基础的外显子捕获检测的关键癌症相关的基因突变在临床肿瘤标本通过大规模并行的“下一代”测序的准备。靶向外显子的测序提供了高吞吐量，低成本，以及深的序列覆盖率的好处，从而产生较高的灵敏度，用于检测低频率突变。

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

detecting-somatic-genetic-alterations-tumor-specimens-exon-capture

Research

JoVE Journal

Biology

19.3K 次观看.  Memorial Sloan-Kettering Cancer Center. 我们描述条形码DNA文库和随后的杂交为基础的外显子捕获检测的关键癌症相关的基因突变在临床肿瘤标本通过大规模并行的“下一代”测序的准备。靶向外显子的测序提供了高吞吐量，低成本，以及深的序列覆盖率的好处，从而产生较高的灵敏度，用于检测低频率突变。 

视频: Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T淋巴细胞艾滋病毒的捕获使用一次性微流体芯片

科研

生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

线性扩增介导的PCR  - 未知侧翼DNA的遗传物质和表征本地化

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.